CN102083990B - Enzymatic decarboxylation by 3-hydroxy alkanoic acid is prepared alkene - Google Patents

Abstract

The present invention relates to generate by biology the method for alkene. It relates to the method for preparing end alkene by the enzymatic decarboxylation of 3-hydroxy alkanoic acid molecule more specifically. The invention still further relates to the microbial strains of enzyme system and use, and the product obtaining.

Description

Enzymatic decarboxylation by 3-hydroxy alkanoic acid is prepared alkene

Brief introduction

The present invention relates to generate through biological process the method for alkene. More specifically, the present inventionRelate to from 3-hydroxy alkanoic acid type molecule and prepare end alkene (particularly propylene, ethene, 1-Butylene, isobutene or iso-amylene) method.

Background of invention

Current many chemical compounds derive from petroleum chemicals. Alkene (for example ethene,Propylene, different butylene or amylene) be applied to plastics industry (for example produce polypropylene orPolyethylene) and the other field of chemical industry and fuel.

The simplest alkene is the core of ethene in industrial organic chemistry: it is to give birth in the worldProduce organic compound the most widely. Especially for producing polyethylene, a kind of main mouldingMaterial. Ethene can be converted into many industrial useful products by (oxidation, halogenation) reactionProduct.

Propylene has similar important function: its polymerization produces plastic material polypropylene.The technology spy of this product aspect drag, density, robustness, deformability and transparencyProperty is beyond challenge. After being invented from 1954, worldwide polypropylene cityField is with regard to sustainable development.

Butylene exists with 4 kinds of forms, and one of them isobutene is methyl tertiary butyl ether(MTBE)(MTBE) part, MTBE is the antiknock dope of motor vehicle fuel. IsobuteneAlso can be used for producing isooctene, after it, can be reduced to isooctane (2,2,4-trimethylPentane); The high combustion/explosion ratio of isooctane becomes for so-called " gasoline "The optimum fuel of engine.

Amylene, hexene and heptene exist in a variety of forms according to position and the configuration of two keys.These products have actual commercial Application, but do not have ethene, propylene or butylene important.

All these alkene are prepared (or from coal or vapour by the catalytic pyrolysis of oil product at presentPrepared by the derivative by Fisher-Tropsch method in oil, for example hexene). ThereforeIts cost natural according to oil price adjustment. In addition, catalytic pyrolysis is followed quite sometimesTechnical difficulty, thereby improve process complexity and production cost.

Except above consideration, the biology preparation of plastics is flourishing fields. StoneThe economy worry that oil price lattice are relevant and the whole world (carbon neutralization (carbon-neutral) product)And the two environmental consideration of region (waste management) has driven sending out rapidly of biological preparationExhibition.

The major families of biological plastics is PHA (PHA). These polymerObtained by the molecule condensation that comprises acidic group and alcohol radical simultaneously. Condensation is passed through acid to next one listThe esterification of the alcohol of body is carried out. This ester bond is not in conventional plastic polymerDirect carbon-carbon bond stable, this has explained that PHA why has several thoughtful some monthsBiodegradability.

PHA family specifically comprises poly 3-hydroxy butyrate (PHB), 3-hydroxybutyrate esterPolymer and polyhydroxybutyrate valerate (PHBV), 3-hydroxybutyrate ester and 3-hydroxylThe alternating polymer of valerate.

PHB is by for example alcaligenes eutrophus (Alcaligenes of several bacterial isolatesesAnd the natural generation of Bacillus megatherium (Bacillusmegaterium) eutrophus).The laboratory that has built the route of synthesis of having integrated substantially guiding PHB or PBA is thinBacterium, for example Escherichia coli. This compound or its polymer can under some laboratory conditionsAccount for bacterium weight up to 80% (people such as WongMS, Biotech.Bioeng.,2008). Attempt the industrial-scale production of PHB in the eighties in 20th century, but at that timeThink that to prepare the cost of this compound by fermentation too high. Plant at genetic modification is (wholeClosed the key enzyme of the PHB route of synthesis existing in producer bacterium) in relevant theseThe project of the direct preparation of compound is being carried out and may only needed lower operation to becomeThis.

Under the overall situation of sustainable development industrial operation of coordinating with geochemical cycle,Need to produce and can be used for the alkane of fuel or synthetic resin precursor or other by biological approachOrganic molecule. First generation bio-fuel is taking the fermenting and producing of ethanol as main because fermentation andStill-process is food-processing industry Already in. The production of second generation bio-fuel is just locatedProbe phase, specifically comprise long-chain alcohol (butanols and amylalcohol), terpene, linear alkane and fatThe production of acid. 2 sections of nearest summaries provide the General Introduction of this area research:The people such as LadyginaN, ProcessBiochemistry, 2006,41:1001; WithWackettLP，CurrentOpinionsinChemicalBiology，2008，21：187。

In the chemical family of alkene, isoprene (2-methyl isophthalic acid, 3-butadiene) beIn polymerization, generate the terpene motif of rubber. Other terpenes may be by chemistry, biological or mixing wayFootpath generates, as for example bio-fuel of enabled production or for the manufacture of plastics. Nearest literary compositionOffer and show that mevalonate pathway is (in the key in a lot of biological steroids biosynthesisMesosome) may be used for effectively producing product (Withers from terpene family with industrial outputThe people such as ST, Appl.Environ.Microbiol., 2007,73:6277).

[2 is single or disubstituted ethene to end alkene: H2C=C (R¹)(R²)] production brightAobvious study less. In little rhodotorula (Rhodotorulaminuta), detect fromIsovaleric acid is to generation (people such as FujiiT., the Appl.Environ. of isobuteneMicrobiol., 1988,54:583), but the efficiency of this conversion is lower than per minute 1,000,000/ mono-or about every day one one thousandth, do not reach commercial Application far away. FukudaH.Deng people (BBRC, 1994,201 (2): 516) illustrated reaction mechanism, wherein related to thinBorn of the same parents' cytochrome p 450 enzyme, it is by reduction oxygenate iron (oxoferryl) base Fe^V=O is to differentValeric acid decarboxylation. In reaction, do not relate to the hydroxylating of isovaleric acid. Isovaleric acid is also that leucine dividesSeparate the intermediate in metabolism. Seem very by the extensive biosynthesis isobutene of this approachImproper, because it need to synthesize and degrade, 1 molecule leucine is to form 1 molecule isobutylAlkene. And the enzyme of catalytic reaction uses ferroheme as confactor, be not suitable for carefullyRecombinant expressed and improvement enzyme parameter in bacterium. Due to all these reasons, under prior artThis approach seems can not serve as very much the basis of commercial development. Can be a small amount of from isoamylOther microorganisms of the natural generation isobutene of acid also have description, but the output obtaining evenOutput than little rhodotorula lower (people such as FukudaH., Agric.Biol.Chem.,1984，48：1679)。

The natural generation of propylene has also been described in these identical researchs: a lot of microorganisms canProduce propylene, but output is still extremely low.

Know for a long time plant can produce ethene (people such as Meigh, 1960, Nature,186:902). According to the metabolic pathway of having illustrated, methionine is the precursor of ethene(Adams and Yang, PNAS, 1979,76:170). The conversion of 2-oxoglutaric acid also hasDescribe (people such as LadyginaN., ProcessBiochemistry2006,41:1001). Because the 4-or the 5-carbochain that before independent ethylene molecule needs, produce,The needed equipment of all these approach and energy are inappropriate, are unfavorable for that it is for lifeThing is produced the commercial Application of alkene.

At the true metabolic precursor thereof S-adenosylmethionine that identifies ethene in plant(SAM) by form 1-amino-cyclopropane-1 carboxylate (ACC) (Adams and Yang,PNAS, 1979,76:170) be converted into the enzyme step of ethene before, in scientific literatureOnce propose other several hypothesis and explained the generation of ethene, de-comprising 3-hydracrylic acidThe decarboxylation of the acrylic acid (H2C=CH-CO2H) of water generates. Warp specifically inferred in some articlesAcrylic acid is converted into the metabolic pathway of ethene from 3-hydracrylic acid, to explain that ethene producesRadioactive tracer research, wherein in plant tissue prepared product, apply¹⁴C markSubstrate: apply Beta-alanine-2-in soybean cotyledon extract¹⁴C (Stinson andSpencer, PlantPhysiol., 1969,44:1217; Thompson andSpencer, Nature, 1966,210:5036), and apply third in banana pulp homogenateAcid-2-¹⁴C (Shimokawa and Kasai, Agr.Biol.Chem., 1970,34 (11): 1640). All these hypothesis relate to 3-hydroxyl in the ethene of metabolism producesPropionic acid and acrylic acid, and do not identify enzymatic activity, finding methionine, SAM and ACC(Hanson and Kende, PlantPhysiology, 1976,57:528 afterwards; AdamsAnd Yang, PNAS, 1979,76:170), these hypothesis just disappear from scientific literature.

Therefore, so far as I know, do not synthesize and for example produce end alkene by microorganism at presentThe effective ways of ethene, propylene, 1-butylene, isobutene, 1-amylene or iso-amylene. ThisA little methods may be avoided using oil product and reduce the cost of producing plastics and fuel. ?After, these methods can be stored carbon with solid form, may have considerable whole world ringBorder impact.

Summary of the invention

The invention describes through bioprocess and carry out the synthetic method of ene compound.

The present invention is based on the design of the novel route of synthesis of end ene compound, described approachBased on the conversion of 3-hydroxy alkanoic acid. The present invention is also based on can be with biology to described conversionThe proof that mode is carried out, by being used enzyme or its variant of decarboxylase type. The present invention canExternal realization in cell free system, or by using microorganism to realize. The present invention also relates toAnd from carbon source, prepare alkene, described carbon source is in particular carbohydrate (particularly grapeSugar), polyalcohol (particularly glycerine), biodegradable polymer (particularly form sedimentPowder, cellulose, poly--3-hydroxy alkanoic acid); Carbon source by microbial conversion for belonging to 3-hydroxylThe metabolic intermediate of base alkanoic acid family, is converted into end alkene afterwards again.

More specifically, the object of this invention is to provide the method for preparing end alkene, its featureBe that described method comprises following steps: under the existence of enzyme with decarboxylase, turnChange 3-hydroxy alkanoic acid.

Another object of the present invention be based on 3-hydroxy alkanoic acid compound as precursor orSubstrate is for the preparation of the purposes of end ene compound.

In specific embodiment of the present invention:

-3-hydracrylic acid is converted into ethene; Or

-3-hydroxybutyrate is converted into propylene; Or

-3-hydroxypentanoic acid is converted into 1-butylene; Or

-3-hydroxyl 3 Methylbutanoic acid (or 3-hydroxyl isovaleric acid) is converted into isobutene; Or

-3-hydroxyl 3 methylvaleric acid is converted into iso-amylene.

The microorganism that the invention still further relates to decarboxylase or produce decarboxylase is from 3-hydroxyl alkaneThe purposes in end ene compound is prepared in acid.

The invention still further relates to composition, it comprises the microorganism, suitable that produces decarboxylaseCulture medium and 3-hydroxy alkanoic acid compound or can be 3-hydroxy alkanoic acid by microbial conversionThe carbon source of compound.

Another object of the present invention relates to biocatalyst, and it comprises 3-hydroxyl alkaneAcid compound decarboxylation is the decarboxylase of end alkene or the microorganism of preparing decarboxylase.

Another object of the present invention relates to the end being obtained by the method for describing as the present inventionEnd ene compound.

Another object of the present invention be separate or purifying there is decarboxylase alsoThe all or part of enzyme that comprises SEQIDNO:6, or there is at least 15% sequence with itThe enzyme of homology.

Another object of the present invention relates to and has decarboxylase and comprise SEQIDThe all or part of enzyme of NO:6, or there is the enzyme of at least 15% sequence homology with itIn the purposes of preparing in end alkene.

Another object of the present invention relates to preparation to be had decarboxylase and comprises SEQThe all or part of enzyme of IDNO:6 or there is the enzyme of at least 15% sequence homology with itMethod, described method is included in to allow to express under the condition of described sequence and cultivates and comprise volumeThe microorganism of the recombinant nucleic acid of the described sequence of code.

Another object of the present invention relates to microorganism, and it comprises coding and has decarboxylase workProperty all or part of enzyme that comprises SEQIDNO:6 or have at least 15% with itThe recombinant nucleic acid of the enzyme of sequence homology.

Definition

As used herein, " 3-hydroxy alkanoic acid " represents to comprise the conduct of 3-hydracrylic acidAny molecule (Fig. 1) of common motif, optional have 1 or 2 alkane on 3 carbonBase replaces. Described alkyl residue or group can be linear or branch. As used herein, term " alkyl (alkoyl) " and " alkyl (alkyl) " have identicalThe meaning is also interchangeable. Similarly, term " residue " and " group " have identical meaningThink also interchangeable. Described in methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group beThe example of alkyl. If 2 alkyl replace difference, 3 carbon become chiral centre. ThisDefinition comprises 2 kinds of chirality forms, even when the one in 2 kinds of forms, and for example R type,Also like this during for the principal mode of natural generation. The example of 3-hydroxy alkanoic acid is shown in Fig. 3. AppointChoosing, alkyl replaces and can be added on 2 carbon, also may become after it chirality (as2 of fruits replace different). Identical, the configuration of 3-hydroxy alkanoic acid substrate in the present inventionComprise all stereoisomers. In preferred mode, the corresponding 3-of 3-hydroxy alkanoic acidThe variant of hydracrylic acid or 3-hydracrylic acid or derivative, wherein 2 on 3 carbonOne of hydrogen atom or 2 hydrogen atoms are replaced by the motif being only made up of carbon and hydrogen atom, instituteState substituent carbon atom number range from 1 to 5, preferably from 1 to 3, for example methyl,Ethyl, propyl group, isopropyl, butyl or isobutyl group. As used herein, suffix " acid(oate) " interchangeable expression Carboxylic Acid Ions (COO-) or carboxylic acid (COOH). It need notIn representing ester. In specific embodiment, 3-hydroxy alkanoic acid represents with following general formula:HO-CO-CH₂-C(R¹)(R²)-OH or O^--CO-CH₂-C(R¹)(R²)-OH。

According to the present invention, " end alkene " represents only (have general formula CnH2n by carbon and hydrogenUnsaturated hydrocarbon) composition molecule, it comprises having of ethene and ethylene derivativeMachine molecule, the latter is by being combined in 2 hydrogen atoms on 2 carbon by linearity or branchAlkyl list or two replacement. End alkene is preferably by general formula H2C=C (R¹)(R²) represent, itsMiddle R¹And R²Independently be selected from the alkyl of hydrogen atom and linearity or branch, described alkyl is excellentChoosing there is 1 to 4 carbon atom, be preferredly selected from 1 to 3 carbon atom. Preferably, at least 1 in 2 substituting groups on 2 carbon of alkene is the alkane of linearity or branchBase. The isoolefine compound that end alkene comprises branch, for example isobutene. According to of the present inventionThe preferred exemplary of end ene compound is specially ethene, propylene, isobutene and iso-amylene (figure4), or 1-butylene and 1-amylene.

As used herein, " carbon source " represents can be used for the substrate of the biology according to the present inventionAny carbon compound. Described term comprise glucose or arbitrarily other hexoses, wood sugar orOther pentoses arbitrarily, polyalcohol is glycerine, sorbierite or sweet mellow wine for example, or polymer exampleAs starch, cellulose or hemicellulose, or for example poly-3-hydroxyl fourth of poly-3-hydroxy alkanoic acidAcid. It can be any substrate that allows growth of microorganism, for example formic acid. It also can beCO₂If this biology can carry out photosynthesis.

As used herein, " restructuring " represents biological artificial genetic modification, by rightThe interpolation of chromosome or chromosome alia gene or regulation and control motif for example promoter, remove or repairDecorations, or by biological fusion, or for example, by adding the carrier of any type, plasmid.Term " recombinant expressed " represents the generation of the protein that relates to genetic modification, preferablyThe protein of preparation for its host's external source or allos, described protein is not naturalBe present in preparation host, or in order to prepare endogenous protein modification or sudden change.

As used herein, " excessively expressing (overexpression) " or " expression excessively(overexpressing) " the sky of expression recombinant expression of proteins and described proteinSo express and compare raising at least 10%, preferred 20%, 50%, 100%, 500% and possibleMore, the microorganism that preferred described protein is originated and express its microorganism notWith. This definition also comprises the situation of the natural expression that does not have described protein.

" cosubstrate (co-substrate) " is the product that is added into enzyme reaction, usesIn improving its special parameter, the most important thing is to improve its activity, described product and the main endThing is with same amount consumption. Therefore cosubstrate must add with the concentration suitable with main substrateEnter reaction. Depend on enzyme, the existence of cosubstrate may be that enzyme reaction is needed.

" confactor " is the product that is added into enzyme reaction, for improvement of its special parameter,The most important thing is to improve its activity, described product does not consume in reaction, therefore only needsTo add to the proportional low concentration of amount of enzyme, therefore described concentration be called as that " catalysis is (denseDegree) ".

" part " of amino acid sequence represents to comprise at least 10, and preferably at least 20,30,The fragment of 40 or 50 continuous described sequence amino acid residues.

" homology " represents the similitude existing between 2 sequences, by described 2The percentage homogeneity of bar sequence is measured.

Compound is often known with several names, formal or common. Here,The preferably generic name of molecule. Therefore:

-" ethene (ethylene) " is for representing ethene (ethene)

-" propylene (propylene) " is for representing propylene (propene)

-" butylene (butylene) " is for representing butylene (butene)

-" isobutene (isobutylene) " is for representing 2-metering system or isobutene(isobutene)

-" amylene (amylene) " is for representing amylene (pentene)

-" iso-amylene (isoamylene) " is for representing 2-methyl butene or iso-amylene(isopentene)

-" propionic acid (propionate) " is for representing propionic acid (propanoicacid)Or propionic acid ion

-" butyric acid (butyrate) " for represent butyric acid (butanoicacid) orButyric acid ion

-" valeric acid (valerate) " for represent valeric acid (pentanoicacid) orValeric acid ion.

Detailed Description Of The Invention

Specifically, the invention provides the method for preparing end alkene, it comprises 3-hydroxylThe enzymatic decarboxylation step of base alkanoic acid compound. The invention still further relates to the decarboxylation of this reaction of catalysisEnzyme, concrete is the enzyme of mevalonic acid diphosphonic acid decarboxylase type, and such as 3-of substrateHydroxybutyric acid, 3-hydroxypentanoic acid, HMB (or 3-hydroxyl isovaleric acid)Purposes with 3-hydracrylic acid. The invention describes the purposes of confactor, described auxiliaryHelp the factor to comprise ethyl diphosphonic acid, propyl group diphosphonic acid, methyldiphosphonic acid, described moleculeAnalog and pyrophosphoric acid. The present invention has also described the purposes of cosubstrate, for example ATPOr other compounds that comprise phosphoric anhydride bonds.

The invention still further relates to carbon source, for example glucose, from full cell by 3-hydroxylThe route of synthesis that base alkanoic acid carries out is directly prepared the purposes of end alkene.

The invention still further relates to biology natural or that modify, its endogenous generation 3-hydroxy alkanoic acidAnd also express the decarboxylase that described 3-hydroxy alkanoic acid is converted into end alkene.

Prepared ene compound, particularly propylene, ethene and isobutene, be plastics andKey molecule in fuel industry, it is the industry from renewable resource by biological approachPreparation has represented great innovation.

Therefore the present invention is establishing according to the novel route of synthesis of end alkene type compoundMeter obtains, the conversion of described approach based on 3-hydroxy alkanoic acid type compound. The present inventionProve that described conversion can be undertaken by biology, by using the enzyme of decarboxylase type, instituteState enzyme and 3-hydroxy alkanoic acid can be converted into end alkene. As shown in Figure 2, described conversionUndertaken by the reaction intermediate with 3-phosphoric acid-hydroxy alkanoic acid structure.

Can (or comprise in addition one or many at the enzyme separating according to step of converting of the present inventionThe enzyme system of kind of confactor) carry out in vitro under existing, or in the microorganism that produces enzymeUnder existence, in culture, carry out.

As described in embodiment 5 herein, under some conditions, can be observed and turnChange the signal to noise ratio (measuring) of approximately 100 times of output in the time not there is not enzyme. It is right to have measuredThe affinity of 3-hydroxyl isovaleric acid (HIV) is about 40mM. It is not immediately clear aobvious like thisHow the enzymatic activity of work obtains: in fact, be familiar with the biochemistry of zymetology theory and practiceScholar knows clearly, enzyme active sites comprise can identify, combination and chemical conversion someThe structural detail of specific substrate. Scientific literature and experimental data instruction, size or electric chargeChange, even minor alteration can cause the repulsion of substrate. Specifically, there is no sciencePrediction can expect that MDP decarboxylase can use the general molecule spy of 3-hydroxy alkanoic acid typeBe not 3-hydroxyl isovaleric acid as substrate, the difference of the latter and mevalonic acid diphosphonic acid is not(molecular weight 118, mevalonic acid diphosphonic acid is 308) only in size, and in skyOn the electric charge of the diphosphonic acid group so existing in substrate mevalonic acid diphosphonic acid.

In a specific embodiment, in reaction, add confactor with in catalysisSpace or electronics complementation are provided in crack. Confactor be advantageously selected from pyrophosphate ion,Methyldiphosphonic acid, ethyl diphosphonic acid or propyl group diphosphonic acid. More generally, confactor isThe compound that comprises phosphoric anhydride motif, it has general formula R-O-PO₂H-O-PO₃H₂, whereinR specifically represents hydrogen atom, and the alkyl of linearity, branch or ring-type, preferably has from 1To 10 or from 1 to 5 carbon atoms, or other unit price organic groups arbitrarily. The present inventionAlso comprise the similar motif of corresponding Medronate monoesters, by general formulaR-O-PO₂H-CH₂-PO₃H₂Representative, wherein phosphoric anhydride is replaced by methylene bridge, its advantageBe not to be hydrolyzed.

In preferred embodiments, transform under cosubstrate exists and carry out, described auxiliaryPreferably comprised the compound of phosphoric anhydride with thing, preferably ATP, rNTP, NTPOr the mixture of several these molecules, polyphosphoric acid or pyrophosphoric acid. Cosubstrate generally existsIn host. But, in another specific embodiment, cosubstrate can be addedAdd in reaction, it is preferably selected from ATP, rNTP, NTP, some rNTP or dNTPMixture, polyphosphoric acid and preferred pyrophosphoric acid, or the compound that comprises phosphoric anhydride (byGeneral formula X-PO in Fig. 2₃H₂Representative).

In a specific embodiment of the present invention, use and produced the micro-of decarboxylaseBiological. In preferred embodiments, microorganism is recombinated, and it produces relatively and producesThe decarboxylase of host's allos. Therefore described method can directly be carried out in culture medium, does not needWant isolated or purified enzyme system. In a kind of mode having superiority especially, use followingMicroorganism: it has the natural or artificial of one or more 3-hydroxy alkanoic acids of endogenous generationPerformance, and also express or cross and express natural or modified decarboxylase, thereby from solutionThe carbon source existing is directly prepared end alkene.

The microorganism that the present invention uses can be prokaryotes or eucaryote, specifically thinBacterium, yeast, plant cell, fungi and mould, zooblast. A concrete realityExecute in scheme, microorganism is bacterium, and concrete is alcaligenes eutrophus and Bacillus megatheriumBacterial strain.

In another specific embodiment, microorganism is the Escherichia coli of restructuring(Escherichiacoli) bacterial strain, its modified with endogenous generation one or more3-hydroxy alkanoic acid, and they are converted into end alkene.

In another specific embodiment, microorganism is the yeast of restructuring, its generation3-hydroxy alkanoic acid is also converted into end alkene by them.

In another specific embodiment, use microorganism produce on the one hand a kind of orMultiple 3-hydroxy alkanoic acid, uses decarboxylase on the other hand, optional by the micro-life of the secondThing is expressed decarboxylase. Optional, the method according to this invention is cultivated and is used 2 simultaneouslyPlant microorganism.

In another specific embodiment, (optional is logical to use whole plant or animalCrossing transgenosis modifies) prepare end alkene from 3-hydroxy alkanoic acid, no matter they are endogenous productsRaw or external source supply.

In another specific embodiment, use following photosynthetic microorganism: it hasNatural or the artificial performance of one or more 3-hydroxy alkanoic acids of endogenous generation, and also cross tableReach natural or modified decarboxylase, thus the CO existing from solution₂Directly preparation endEnd alkene. Preferably, described microorganism is photosynthetic bacterium or micro-algae.

The invention still further relates to the microorganism of describing hereinbefore and prepare end alkylene with themThe purposes of compound.

As described below, method of the present invention can be carried out under microaerophilic condition.

In addition, in preferred embodiments, method is at collection degassed end from reactionUnder the existence of the system of end alkene gas, carry out.

As used herein, decarboxylase represents will have the 3-hydroxyl of n carbon atomBase alkanoic acid is converted into any enzyme of the end alkene with n-1 carbon atom. As at Fig. 2Shown in, method of the present invention is preferably entered through 3-phosphoric acid hydroxy alkanoic acid reaction intermediateOK, and use enzyme advantageously there is decarboxylase and phosphorylase activity.

In a specific embodiment, decarboxylase is mevalonic acid diphosphonic acid (MDP)Member in the superfamily that the system of decarboxylase (enzyme name EC4.1.1.33) occurs,That is to say, by natural or synthetic gene coding, optional can catalysis Fig. 2 in instituteNatural or the artificial enzyme of the reaction of showing.

MDP decarboxylase is the enzyme that participates in Biosynthesis of cholesterol. From many biological bagsDraw together in animal, fungi, yeast and some bacteriums and isolate described enzyme. They also can be by someExpression of plants (people such as Lalitha, 1985). Cloning and sequencing many codings thisThe gene of enzyme. These enzymes are generally made up of 300 to 400 amino acid, use ATP conductCosubstrate, ATP is converted into ADP and inorganic phosphate in reaction. Phosphate group is from ATPMolecular transfer, to the tertiary alcohol of mevalonic acid diphosphonic acid, discharges ADP. At 3-oh group phosphorusCancellation phosphate after the reaction intermediate of acidifying discharges iso-amylene Jiao under physiological conditionPhosphoric acid (Fig. 2).

Resolve the three-dimensional structure of some enzymes of Liao Ci family. Up to the present, to this familyThe research that family's enzyme carries out is relatively less, only at the biosynthesis pathway of accurately describing cholesterolIn studied these enzymes. On the other hand, so far as I know, also do not study this enzyme from itConversion become industrial catalyst in natural function.

At SEQIDNO:1 to having provided in the sequence of SEQIDNO:16 from notWith some examples of biological MDP decarboxylase.

Therefore, in preferred embodiments, the enzyme of use is decarboxylase, and it is preferredComprise and be selected from SEQIDNO:1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 or 16 amino acid sequence, or have at least with one of described sequence15% sequence homology also retains the sequence of decarboxylase. Preferred enzyme advantageously withSEQIDNO：1，2，3，4，5，6，7，8，9，10，11，12，13，14，One of basic (primary) sequence of 15 or 16 has at least 50% sequence homology,Preferably at least 80%, preferred at least 85%, even preferred at least 90,95,96,97,98 or 99% homology. Determine that sequence homology percentage can pass through not TongfangMethod and by for example CLUSTAL method of software program well known by persons skilled in the art orBLAST, and derivative software, or use sequence comparison algorithm for example Needleman andWunsch (J.Mol.Biol., 1970,48:443) or Smith and Waterman (J.Mol.Biol., 1981,147:195) described.

The representative of the preferred decarboxylase of the present invention is the enzyme with SEQIDNO:6 sequenceWith any enzyme with it with significant sequence homology. Preferred enzyme advantageously with SEQThe basic sequence of IDNO:6 has at least 50% sequence homology, and preferably at least 80%,Preferred at least 85%, even preferred at least 90,95,96,97,98 or99% homology. Described enzyme is from have a liking for bitter ancient bacterium (Picrophilustorridus) gramGrand and prepared by the recombination method in the scope of the invention. As shown in Example, this enzymeEspecially effectively preparation is according to end ene compound of the present invention. This enzyme and preparation and itsAlso be object of the present invention as the purposes of catalyst. Concrete, an order of the present inventionBe to comprise all or part of decarboxylase of SEQIDNO:6 or and SEQIDNO:6The enzyme with significant sequence homology, preferred at least 15% homology is being prepared endPurposes in ene compound. Significant sequence homology refers to and uses above-mentioned algorithm to detectSequence homology, preferably higher than 15% sequence homology. And have a liking for bitter ancient bacterium and haveThe biology of nearest phylogenetic relationship can be prepared the most similar to SEQIDNO:6MDP decarboxylase, described biology, for for example Ferroplasmaacidarmanus, is had a liking for acidPyrogen body (Thermoplasmaacidophilum), hot volcanic substanceAnd Picrophilusoshimae (Thermoplasmavolcanium). For example,Thermoplasma acidophilum's (AC Q9HIN1) MDP decarboxylase and SEQIDNO:6 have38% sequence homology; MDP decarboxylase and the SEQID of hot volcanic substance (Q97BY2)NO:6 has approximately 42% sequence homology. These are considered more specifically in the present inventionThe purposes of MDP decarboxylase.

Can select natural or synthetic according to the ability of end alkene of the present invention according to enzyme preparationThe enzyme of other decarboxylase types. Therefore, select test to comprise the enzyme of purifying or generationThe microorganism of enzyme contacts with reaction substrate, and measures the generation of end ene compound. TheseTest is described in embodiment part, has wherein detected and has exceeded 60 kinds of different enzymes.

The enzyme using can be natural, any decarboxylase preparation or artificial optimization. ToolBody, advantageously use one or more 3-hydroxy alkanoic acids are had and optimize active taking offCarboxylic acid.

Can be from reference to decarboxylase (natural or self be synthetic or optimize) preparationOr selection enzyme, by protein engineering for example random mutagenesis, a large amount of mutagenesis, fixed pointIn mutagenesis, DNA reorganization, synthetic reorganization, body, evolve or complete synthetic gene.

Thus, one object of the present invention also relates to preparation for 3-hydroxy alkanoic acidSubstrate has the method for the enzyme of decarboxylase, and described method comprises following steps: to enzymeProcess in source, and selection has enhancing for described substrate compared with untreated enzymeThe enzyme of performance.

Therefore the enzyme using in the present invention can be natural or synthetic, by chemistry, rawThing is learned or genetic method preparation. It also can be chemical modification, for example with improve its activity,Resistance, specificity, purifying or be fixed on holder.

The invention is characterized in decarboxylase, particularly natural or modify MDP decarboxylase3-hydroxy alkanoic acid is converted into the purposes in end alkene.

The natural substrate of MDP decarboxylase is mevalonic acid diphosphonic acid, and it does not belong to 3-hydroxylThe range of definition of alkanoic acid.

In Fig. 2 B, describe MDP decarboxylase and used general that multiple 3-hydroxy alkanoic acid carries outReaction. Think that these reactions directly obtain end alkene with a step.

In first embodiment, use natural or restructuring, purifying or not purifiedEnzyme 3-hydroxy alkanoic acid is converted into end alkene. For this reason, under existing, substrate allowingUnder the activated physical and chemical condition of enzyme, hatch enzyme preparation, allow to hatch carry out sufficiently longTime. Hatching after end, the existence of optional measurement end alkene, by this area skillThe for example gas chromatography of any detection system that art personnel are known or colorimetric test are measured alkene and are producedThe formation of thing or free phosphoric acid, or the disappearance of measuring 3-hydroxy alkanoic acid substrate or ATP.

In preferred embodiments, interpolation confactor is natural anti-with best simulationShould. In fact, the general corresponding MDP fragment of the structure of 3-hydroxy alkanoic acid, therefore enzyme-In Binding Capacity, in catalysis crack, reserve empty huge space. Fill this by confactorSpace is to replace the disappearance part of substrate, and object is the most closely to simulate MDP molecule.Because confactor is not modified in reaction, therefore it only adds with catalytic amount. InsteadAnswering substrate is in the situation of 3-hydracrylic acid, and complementary confactor is propyl group diphosphonic acid.In the situation that reaction substrate is 3-hydroxybutyrate or HMB, complementationConfactor be ethyl diphosphonic acid. 3-hydroxypentanoic acid or 3-hydroxyl at reaction substrateIn the situation of-3 methylvaleric acid, complementary confactor is methyldiphosphonic acid. These are notSame molecule is shown in Fig. 5. Sometimes, the complementary confactor of a reaction may be rightThe reaction of another substrate has positive role. General, confactor can be arbitrarily and comprisesThe molecule of phosphoric anhydride, therefore has general formula R-PO₂H-O-PO₃H₂, wherein R specifically H,Linear, branch or cyclic alkyl, or other unit price organic groups arbitrarily. The present invention also wrapsThe similar motif of drawing together corresponding Medronate monoesters, it has general formulaR-O-PO₂H-CH₂-PO₃H₂, wherein phosphoric anhydride is replaced by methylene bridge, it is advantageous that notBe hydrolyzed.

More generally, confactor can be monophosphate or or even above molecule without phosphoric acidAnalog, or produce by provide space or electric charge complementation can improve reaction in enzymatic siteAny other molecules of amount.

In a specific embodiment, in reaction, add cosubstrate. Described auxiliaryBe can be ATP with thing, i.e. the natural cosubstrate of MDP decarboxylase, or any rNTP(ribonucleotide triphosphate) or dNTP (deoxyribonucleoside triphosphate) or rNTP orAny mixture of dNTP, or pyrophosphoric acid, or other polyphosphoric acid, or other comprise phosphorusAny molecule (X-PO in Fig. 2 of anhydride group₃H₂)。

In preferred embodiments, for 3-hydroxy alkanoic acid is converted into end alkene,Used with have the native enzyme of decarboxylase, particularly with corresponding SEQIDNO:1There is at least 15% sequence homology to one of enzyme of 16 sequences, preferably at least 30%,50% and even preferred at least 80,90,95,96,97,98 or 99% sequence is sameThe enzyme of source property. Concrete, enzyme can be modified, by SEQIDNO:1's to 16Any other decarboxylase transformations of one of enzyme or other Identification of The Origins obtain. These enzymesMay lose its MDP decarboxylase, particularly in the genetic modification in laboratory,Also may in natural evolution, (can be described as in this case the MDP decarboxylase of degeneration),But retain or improved the activity for one or more 3-hydroxy alkanoic acid types of molecules.The generation of the variant to these higher enzymes of described substrate activity likely improves according to thisThe reaction yield of invention. For example, wild type MDP decarboxylase is for 3-hydroxy alkanoic acidReactive not necessarily the best. Can use any means well known by persons skilled in the artPrepare and select these variants, described method is random mutagenesis, direct mutagenesis, a large amount of for exampleIn mutagenesis, DNA reorganization or body, evolve.

Feature of the present invention is also that artificial enzyme is being converted into 3-hydroxy alkanoic acid completelyPurposes in end alkene, described enzyme is by designing and prepare the synthetic gene of the brand-new enzyme of encodingObtain, by using or do not use the given data design of MDP decarboxylase.

Another object of the present invention is to have decarboxylase and comprise SEQIDNO:6Enzyme all or part of separation or purifying.

Another object of the present invention relates to and has decarboxylase and comprise SEQIDNO:6 all or part of enzymes or have and preparing end alkene as the enzyme of above-mentioned sequence homologyIn purposes. In a variant, sequence can further comprise other residue, for exampleHistidine-tagged at N-end.

Another object of the present invention relates to preparation to be had decarboxylase and comprises SEQThe enzyme that IDNO:6 is all or part of or there is the side of the enzyme of homology with above-mentioned sequenceMethod, described method is included under the condition that allows to express described sequence and cultivates and comprise coding instituteState the microorganism of the recombinant nucleic acid of sequence. In this, the invention describes except naturalNucleic acid (SEQIDNO:19) nucleic acid in addition, it has in bacterium, spyNot not in Escherichia coli, express the enzyme of SEQIDNO:6 and the sequence (SEQ that optimizesIDNO:17). This nucleic acid with the nucleic acid of optimizing arbitrarily (compared with wild-type sequenceCan in expression, obtain at least 30% improvement) be one object of the present invention.

Another object of the present invention relates to and comprising coding and have decarboxylase and comprisingThe enzyme that SEQIDNO:6 is all or part of or there is the enzyme as above-mentioned sequence homologyThe microorganism of recombinant nucleic acid. Microorganism is bacterium, yeast or fungi preferably. ThisBright also relating to, comprises coding according to any plant of the recombinant nucleic acid of decarboxylase of the present inventionOr non-human animal.

In one embodiment, MDP decarboxylase uses with by 3-with the form of purifyingHydroxy alkanoic acid is converted into end alkene. But the method cost is high, because enzyme and substratePreparation and purifying cost are very high.

In another embodiment, MDP decarboxylase is present in the extract of non-purifyingIn reaction, or with the form of non-cracking bacterium, thereby save the cost of protein purification.But the cost that the method is relevant is still very high, because the cost of preparation and purifying substrateHigh.

In another embodiment of the invention, method is used the biology of living to prepare enzyme,Transform by enzyme. Therefore described invention is characterised in that one or more 3-of preparation[richness of for example modifying to produce described product through laboratory is supported for the bacterial isolates of hydroxy alkanoic acidAlcaligenes and Bacillus megatherium, or coli strain] genetic engineering modify, fromAnd making described bacterial isolates cross expression decarboxylase, described enzyme preferably derives from and is different from placeThe biology of main microorganism, and can directly generate one or more end alkene. Genetic modification canComprise decarboxylase gene is integrated into chromosome, the enzyme on expression plasmid, described plasmid bagBe contained in the promoter of enzyme coded sequence upstream, promoter and coded sequence preferably derive fromDifferent biological, or any additive method well known by persons skilled in the art. Optionally, canTo select other bacteriums or yeast, they may have specific advantages. For example, can useFor example saccharomyces cerevisiae of yeast (Saccharomycescerevisiae), has a liking for extreme bacteriaFor example thermus thermophilus (Thermusthermophilus), or from for example clostridium(Clostridiae) anaerobic bacteria of family, micro-algae or photosynthetic bacteria. For optimumPreparation 3-hydroxy alkanoic acid, is converted into end alkene after it, and bacterial strain also can be through hereditary workJourney is modified, by evolving in vitro recombination or directed body.

In one embodiment, the inventive method is characterised in that such as glucose of carbon sourceTo the conversion of 3-hydroxy alkanoic acid, described Primary product is converted into secondary species afterwards,End alkene. The different step of described method shows in Fig. 6.

In a specific embodiment, the invention is characterized in that poly (hydroxyalkanoate) extremelyThe conversion of 3-hydroxy alkanoic acid, by being used enzyme or suitable physico-chemical process, Zhi HousuoState Primary product and be converted into secondary species, be i.e. end alkene. Optional, poly (hydroxyalkanoate) isProduced by plant, the metabolic pathway of described plant is modified so that it produces high yieldPoly (hydroxyalkanoate).

In a specific embodiment, the present invention comprises the CO from atmosphere₂Or peopleWork is added into the CO of culture medium₂Prepare the complete method of product. The method of described invention existsCan carry out photosynthetic biology for example implements in micro-algae.

In these embodiments, being characterised in that in addition from cultivate of method of the present inventionThe take-back model of degassed product. In fact, short end alkene, particularly ethene, thirdAlkene, butylene isomer exist with gaseous state under room temperature and atmospheric pressure. Method of the present invention because ofThis does not need product extracting from fluid nutrient medium, and this step is worked as conventionally with commercial scaleWhile carrying out, cost is very high. Finding time and storage and possible follow-up of hydrocarbon gasPhysical separation and chemical conversion can be entered by any means well known by persons skilled in the artOK.

In a specific embodiment, method of the present invention also inclusion test is present inAlkene (particularly propylene, ethene and isobutene) in gas phase. Object compound is at airOr existence in other gaseous environments, exist even in a small amount, can detect by multiple technologies,Particularly use infrared ray or flame ionization to detect by gas chromatography system, or and mass spectrumCoupling.

In a specific embodiment, the end alkene obtaining is condensation, Zhi HourenThe use technology well known by persons skilled in the art of choosing is through reducing to prepare the more alkene of long-chainOr the alkane of long-chain more. Concrete, isobutene can be used for synthetic isooctane: successfully carry out thisThe detailed description of catalysis process of reaction.

In a specific embodiment, method relates under standard culture condition(30-37 DEG C under 1 atmospheric pressure, in the oxybiontic fermentation tank of permission bacterium)Or for example, under non-standard condition (, the higher temperature of corresponding thermophilic condition of culture)Cultivate microorganism.

In a specific embodiment, microorganism is cultivated under microaerophilic condition,The air capacity that restriction is injected is to minimize the residual oxygen of gaseous effluent that comprises alkeneGas concentration.

Other aspects of the present invention and advantage will be described in following examples, and embodiment isProvide for illustrative purposes, instead of in order to limit.

Brief description of the drawings

Fig. 1: 3-hydracrylic acid motif

Fig. 2: mevalonic acid diphosphonic acid is through the decarboxylation of MDP decarboxylase---general activity

Fig. 3: the example of 3-hydroxy alkanoic acid

Fig. 4: MDP decarboxylase is in the purposes of preparing in end alkene

Fig. 5: in reaction for the complementary structure object in catalytic site spendable auxiliary because ofSon

Fig. 6: the complete method of preparing alkene from glucose

Fig. 7: the chromatography of the enzyme reaction of carrying out in No. 1 condition of embodiment 4

Fig. 8: the lauryl sodium sulfate of crossing expression and purification step of SEQIDNO:6 enzyme is poly-Acrylamide gel electrophoresis (SDS-PAGE)

1. mark

2. the culture before induction

3. lysate

4. the component not absorbed by post

5. the cleaning component of post

6. the enzyme of purifying, molecular weight 36.8kDa

Gas chromatography/mass spectrum (GC/MS) chromatographic analysis that Fig. 9: HIV to IBN transforms

1 and 2: the corresponding negative control that does not contain the background noise of enzyme.

3 and 4: the reaction under SEQIDNO:6 enzyme exists

Figure 10: the ratio that IBN produces in the situation that existing and do not have ATP

Figure 11: have and do not exist Mg²⁺Situation under IBN produce ratio

Figure 12: according to the enzymatic activity of temperature. Ratio: relative background in the situation that there is enzymeIBN formation volume

Figure 13: according to the generation of the IBN of HIV concentration of substrate

Figure 14: the measurement of peak optimization reaction and with the comparison of background. By gas chromatography flameIonization detection is measured.

Figure 15: by the improved expression of optimization of nucleotide sequence of coding SEQIDNO:6Level

M road: molecular weight marker

1,2,3 roads: natural nucleotide sequence

(1) at the cell pyrolysis liquid of purification column loading, soluble constituent

(2) be not retained in the lysate component on purification column

(3) elution fraction: 10 μ g purifying enzymes

4,5,6 roads: the nucleotide sequence of optimization

(4) at the cell pyrolysis liquid of purification column loading, soluble constituent

(5) be not retained in the lysate component on purification column

(6) elution fraction: 10 μ g purifying enzymes

Embodiment

Embodiment 1: the clone of several MDP decarboxylases and expression

The gene of coding saccharomyces cerevisiae MDP decarboxylase is synthetic by overlapping oligonucleotidesAnd be cloned in the pET plasmid (Novagen) that can express in bacterium. Described matterAfter grain, be converted into bacterial isolates BL21 (Invitrogen) by electroporation. At bagContaining ruling bacterium in 37 DEG C of cultivations on the culture dish of ampicillin. Second day, withMachine is selected a bacterial clump and is inoculated in the LB culture medium that 50ml comprises ampicillinIn. Culture is cultivated and is rocked for 24 hours simultaneously, and centrifugal culture afterwards, by ultrasonicRipple is processed cracking bacterium, prepares gross protein extract. Loading decile in running gelThe protein extract of the extract of sample and the identical bacterial strain of unconverted, and molecular weightMark. The corresponding road of bacterial strain that transforms is containing the independent band of the 30kD that has an appointment, and it is corresponding to eggThe expection size of white matter does not have described band in the road of loading unconverted bacterium.

Embodiment 2: measure the activity of protein extract to HMB

HMB (Sigma, numbering 55453, β-hydroxyisovaleric acid by name)Suspend with 10g/l concentration. Mevalonic acid diphosphonic acid is by mevalonolactone and other examinationsAgent (Sigma) is synthesized by conventional method and is resuspended with 10g/l concentration.

Prepare 6 chromatography tubules. In each tubule, add and contain 50mMBistris/HCl, 1mM dithiothreitol (DTT), 10mMMgCl₂With 50 of 5mMATPμ L buffer solution.

Tubule 1 and 4: add 5 μ l water (without substrate).

Tubule 2 and 5: add 5 μ l mevalonic acid diphosphonic acid preparations (positive control).

Tubule 3 and 6: add 5 μ l3-hydroxy-3-methyl butyric acid (HIV) preparations.

Tubule 1,2 and 3: add 5 μ l water (without enzyme).

Tubule 4,5 and 6: add the enzyme preparation of describing in 5 μ l embodiment 1.

Tubule is with diaphragm seal and press pleat. All tubules are hatched 4 hours to 3 at 37 DEG CMy god.

After hatching, use gas syringe to collect the gas in each tubule, pass through gas phaseCO in laminar analysis measurement sample₂Concentration. Tubule 5 has very high CO₂Concentration is littleThe CO of low concentration in pipe 6, also detected₂, indicator enzyme preparation is to 3-hydroxy-3-methylThe remarkable reaction of butyric acid.

Detect and measure tubule 6 gas with infrared ray or flame ionization by gas chromatography afterwardsThe existence of the isobutene in body sample.

Embodiment 3: use confactor to optimize reaction condition

Carry out the same reaction of describing as in the tubule 6 of embodiment above, but at oneIn sample, add (synthesizedtoorder) ethyl two synthesizing as requestedPhosphoric acid is as confactor.

Use in this embodiment 3 tubules. First comprises above institute in embodiment and retouchesBuffer solution, ATP and the enzyme extract of the amount of stating. Second tubule comprises identical composition,But comprise in addition the HMB of the amount described in embodiment above. ?In the 3rd tubule, except HMB, also comprise 10 μ l10mg/lEthyl diphosphonic acid.

As embodiment above, detect and survey with infrared ray or flame ionization by gas chromatographyThe formation of amount isobutene. Find in the time that ethyl diphosphonic acid exists, the isobutene amount of generation withThe time of is obviously higher.

Embodiment 4: screening enzyme library

Obtain coding from the library of 63 genes of the enzyme of MDP decarboxylase family inspectionSurvey its to HIV the activity as substrate.

Clone, Bacteria Culture and protein expression

Clones coding mevalonic acid diphosphonic acid (MDP) the decarboxylase EC4.1.1.33 of familyGene, eukaryotic gene is cloned into pET25b carrier (Novagen), protokaryon sourceGene cloning to pET22b carrier (Novagen), at immediately first sulphur ammonia of N-endAfter acid initiation codon, add 6-histidine-tagged. By these carriers of heat shockTransformed competence colibacillus e. coli bl21 (DE3) cell (Novagen). Cell is in 30 DEG CComprising 0.5M sorbierite, 5mM betaine, 100 μ g/ml ampicillinsIn TB culture medium, rocking (160rpm) cultivates until the OD of 600nm reaches 0.8 HeBetween 1. Add afterwards isopropyl-β-D-thiogalactoside (IPTG) to final concentration1mM, spend the night at 20 DEG C (approximately 16 hours) continue protein expression. By 4 DEG C,Centrifugal 20 minutes collecting cells of 10,000rpm, precipitation is frozen in-80 DEG C.

Lysis

1.6g cell thaws on ice and is resuspended in 5ml and comprises 300mMNaCl, 5mMMgCl₂, 1mMDTT 50mMNa₂HPO₄PH8. Add 20 microlitre lysonase(Novagen). Cell, in incubated at room 10 minutes, is put back to 20 minutes on ice afterwards.By process 3x5 minute cell lysis completely with ultrasonic wave in 0 DEG C of ultrasonic water bath;Stir evenly sample in the pulse spacing. Afterwards by 4 DEG C, centrifugal 20 minutes of 10,000rpm is clearClear bacterial extract.

Protein purification and concentrated (PROTINO kit)

The bacterial lysate loading of clarification is to PROTINO-1000Ni-IDA post(Macherey-Nagel) to carry out the absorption of 6-histidine tagged protein. Clean postAnd comprise 300mMNaCl, 5mMMgCl with 4ml₂，1mMDTT，250mMThe 50mMNa of imidazoles₂HPO₄PH8 wash-out object enzyme. Eluent is afterwards at AmiconUltra-410kDa pipe (cell) is concentrated into the end of 250 μ l in (Millipore)Volume. By Bradford standard measure protein.

Enzyme reaction

Under the buffer solution 2 kind experimental conditions different with reaction pH, detect the enzyme of expecting anti-Answer (HMB, or the conversion of 3-hydroxyl isovaleric acid or HIV).

No. 1 experimental condition:

100mM citric acid

10mMMgCl₂

10mMATP

20mMKCl

200mMHIV

Final pH is adjusted to 5.5

No. 2 experimental conditions:

100mMTris-HClpH7.0

10mMMgCl₂

10mMATP

20mMKCl

200mMHIV

Final pH is adjusted to 7.0

Enzyme is added to reactant mixture. Because protein output difference, in different samplesAdd scope in 0.01 to 1mg/ml enzyme amount. Parallel carry out not containing the contrast of enzyme anti-Should.

In 2ml tubule (Interchim), carry out 1ml reaction, and with teflon/Silica/teflon barrier film (Interchim) sealing. Reaction hatches 72 at 37 DEG CHour, without rocking.

The analysis of reaction

Deposit with equipping in the syringe collecting reaction that can not reply (no-return) deviceGas. By gas chromatography (GC) associating mass spectrum (MS) analytical gas sample.Instrument is in advance with a series of isobutylene concentration calibrations.

Post: BPX5 (SGE)

GC/MS：MSD5973(HP)

Each chromatography is obtained to 3 main peaks, first corresponding air, second corresponding water,The 3rd corresponding isobutene. In 63 enzymes of preparation and determination methods, in primary dcreening operation, identifyGo out 11 potential candidate's enzymes. Some in these candidate's enzymes are used arrow mark in Fig. 7Illustrate. Their qualification shows below, and their sequence is SEQIDNO:6 to 16(not showing histidine-tagged).

Candidate's enzyme 1:SEQIDNO:7

Genebank numbering: CAI97800.1

Swissprot/TrEMBL numbering: Q1GAB2

Microorganism: bacillus acidificans longissimus Bulgaria subspecies (LactobacillusDelbrueckiisubsp.Bulgaricus) (strains A TCC11842/DSM20081)

Candidate's enzyme 2:SEQIDNO:8

Genebank numbering: CAJ51653

Swissprot/TrEMBL numbering: Q18K00

Microorganism: HaloquadratumwalsbyiDSM16790

Candidate's enzyme 3:SEQIDNO:9

Genebank numbering: ABD99494.1

Swissprot/TrEMBL numbering: Q1WU41

Microorganism: Lactobacillus salivarius saliva subspecies (LactobacillussalivariusSubsp.Salivarius) (bacterial strain UCC118)

Candidate's enzyme 4:SEQIDNO:10

Genebank numbering: ABJ57000.1

Swissprot/TrEMBL numbering: Q04EX2

Microorganism: drinks wine coccus (Oenococcusoeni) (bacterial strain BAA-331/PSU-1)

Candidate's enzyme 5:SEQIDNO:11

Genebank numbering: ABJ67984.1

Swissprot/TrEMBL numbering: Q03FN8

Microorganism: Pediococcus pentosaceus (Pediococcuspentosaceus) ATCC25745

Candidate's enzyme 6:SEQIDNO:12

Genebank numbering: ABV09606.1

Swissprot/TrEMBL numbering: A8AUU9

Microorganism: lattice are stepped on streptococcus (Streptococcusgordonii) (bacterial strainChallis/ATCC35105/CH1/DL1/V288)

Candidate's enzyme 7:SEQIDNO:13

Genebank numbering: ABQ14154.1

Swissprot/TrEMBL numbering: A5EVP2

Microorganism: plethora artiodactyl shape bacterium (Dichelobacternodosus) VCS1703A

Candidate's enzyme 8:SEQIDNO:14

Genebank numbering: EDT95457.1

Swissprot/TrEMBL numbering: B2DRT0

Microorganism: streptococcus pneumonia (Streptococcuspneumoniae)CDC0288-04

Candidate's enzyme 9:SEQIDNO:15

Genebank numbering: AAT86835

Swissprot/TrEMBL numbering: Q5XCM8

Microorganism: micrococcus scarlatinae (Streptococcuspyogenes) serotypeM6(ATCCBAA-946/MGAS10394)

Candidate's enzyme 10:SEQIDNO:6

Genebank numbering: AAT43941

Swissprot/TrEMBL numbering: Q6KZB1

Microorganism: have a liking for bitter ancient bacterium (Picrophilustorridus) DSM9790

Candidate's enzyme 11:SEQIDNO:16

Genebank numbering: AAV43007.1

Swissprot/TrEMBL numbering: Q5FJW7

Microorganism: lactobacillus acidophilus (Lactobacillusacidophilus) NCFM

The isobutene (IBN) of observing highest level in candidate's enzyme 10 produces, pureChange from the SEQIDNO:6 decarboxylase of having a liking for bitter ancient bacterium. Retaining this enzyme is used for into oneStep characterizes.

The sign of embodiment 5:SEQIDNO:6 enzyme

Purification of Recombinant enzyme as described in Example 4. The result of showing in Fig. 8 showsThe purity of the enzyme in final protein example is approximately 90%.

Confirm the activity of the enzyme separating. Reaction is carried out in following condition:

100mMTris-HClpH7.0

10mMMgCl₂

10mMATP

20mMKCl

250mMHIV

Final pH is adjusted to 6.0

3mg/ml enzyme

Hatch after 72 hours at 30 DEG C, by GC/MS measuring-signal. The results are shown inFig. 9. Under enzyme exists, the generation of background noise IBN has herein improved approximately 2.3 relativelyDoubly. Consistent with organic chemistry document of the background noise herein of observing, is presented atIn the aqueous solution and at the temperature of approximately 100 DEG C, the decarboxylation slowly of 3-hydroxyl isovaleric acid is tertiary fourthAlcohol, its partial dehydration is isobutene, in accordance with the balance that is conducive to tert-butyl alcohol formation(Pressman and Luca, J.Am.Chem.Soc.1940).

The effect of ATP cosubstrate

Testing conditions

100mM citric acid

50mMKCl

10mMMgCl₂

200mMHIV (to be specified)

The enzyme of 1mg/ml purifying

pH5.5

Hatch 72 hours at 30 DEG C

Condition	ATP final concentration	Enzyme
			1	0mM	0mg/ml
2	0mM	1mg/ml
			3	10mM	0mg/ml
4	10mM	1mg/ml

Result in Figure 10 only shows observes enzymatic activity under cosubstrate ATP exists.Other molecules, the molecule that particularly comprises phosphoric anhydride bonds also for this reason the effective auxiliary of enzyme usedThing.

Mg ²⁺ The effect of confactor

Testing conditions

100mM citric acid pH5.5

50mMKCl

10mMATP

200mMHIV (to be specified)

pH5.5

The enzyme of 1mg/ml purifying

Hatch 72 hours at 30 DEG C

Condition	MgCl2Final concentration	Enzyme
			1	0mM	0mg/ml
2	0mM	1mg/ml
			3	5mM	0mg/ml
4	5mM	1mg/ml

Result in Figure 11 is presented at Mg²⁺Ion exists lower enzymatic activity to improve. OtherIon, particularly other divalent ions, also alternative Mg²⁺Ion or and Mg²⁺Ion altogetherWith using as confactor.

According to the enzymatic activity of temperature

Testing conditions

100mM buffer solution

50mMKCl

10mMATP

200mMHIV (to be specified)

The enzyme of 1mg/ml purifying

Under different temperatures, hatch 72 hours

Result in Figure 12 is presented at the heat of enzyme appropriateness under the Optimal Temperature of approximately 50 DEG C and livesChange.

According to the activity of pH

Testing conditions

100mM buffer solution

50mMKCl

10mMATP

200mMHIV (to be specified)

The enzyme of 1mg/ml purifying

Hatch 72 hours at 30 DEG C

In the time that being 5.5, the pH of 100mM citric acid obtains optimal conditions.

Enzyme parameter

In aforementioned condition, detect substrate scope at 50 DEG C under hatching. The Km of enzyme is for approximately40mMHIV。

The optimization of reaction condition

Study optimum reaction condition, retained following condition:

100mM citric acid

50mMKCl

40mMATP

200mMHIV

1mg/ml enzyme

Hatch 48 hours at 50 DEG C

As shown in Figure 14, the ratio of the relative background noise of signal is approximately 100.

Embodiment 6: have a liking for the optimization of bitter ancient bacterium MDP decarboxylase at expression in escherichia coli

Initial expression in Escherichia coli is very low, because before purifyingOn SDS-PAGE, be difficult to see band. Usehttp://genomes.urv.es/OPTIMIZER/On " Optimizer " program baseMeasure native sequences for Bacillus coli expression in the method for Sharp and Li (1987)Codon optimized index (CAI). The value obtaining only has 0.23, is illustrated in Escherichia coliMiddle protein expression low-level. Generate coding same protein but comprised and be more suitable forThe sequence of the codon of expression in escherichia coli. This sequence has 0.77 CAI, fromOptimum 1 nearer. Native sequences and optimization are shown in SEQIDNO:17 (bagContaining the histidine-tagged optimization of having a liking for bitter ancient bacterium (AAT43941) MDP decarboxylase)And SEQIDNO:19 (comprises histidine-tagged bitter ancient bacterium (AAT43941) MDP that has a liking forThe native sequences of decarboxylase).

Optimization is connected and is synthesized and be cloned into pET25 expression vector by oligonucleotides.According to aforesaid experimental program, carrier is being transformed into e. coli bl21 (DE3) bacterial strainAfter induction, as aforementioned preparation, protein purification and analyze on gel. Right comparison,Native sequences is carried out to identical experimental program.

The relatively expression of candidate's enzyme 224, uses native sequences or at large intestine barIn bacterium, express the sequence of optimizing.

Not impure on gel of the protein that result in Figure 15 shows corresponding optimization geneIn the lysis fluid passage of changing, (road 4) is obviously visible, shows that the expression of highly significant is carriedHigh. Optimization gene is also higher in the purity level of protein after purification step.

In rough lysate, measure active. In the rough cracking of corresponding natural acid sequenceIn liquid, can't detect activity. Improved after protein expression, obtain there is improvement sequenceThe rough lysate of (optimizing clone 224) has been shown this activity now.

Reaction medium below using in this detects:

Reaction medium

?50DEG C hatch 2 days.

Result

No. 1 condition: the lysate of optimizing clone 224

No. 2 conditions: the lysate (empty pET plasmid) of clone GB6

Embodiment 7: from HMB synthetic isobutene be converted into isooctaneMethod

In 1 liter of volume, carry out the reaction identical with tubule 3 in embodiment 3, at dressIn the standby fermentation tank of gas extraction system, carry out. The existence of recombinase causes 3-hydroxyl-3-Methylbutanoic acid is to the conversion of isobutene, and isobutene is natural degassed, and by being positioned on fermentation tankThe gas extraction system of portion reclaims. After isobutene by Amberlyst35wet orThe catalysis addition of 36wet resin (Rohm and Haas) is to prepare isooctene. By catalysisIsooctene is reduced to isooctane by hydrogenation.

Embodiment 8: transformation enzyme is to improve the validity to substrate

Use random mutagenesis technology to set up and comprise the gene of describing in thousands of kinds of embodiment 1Mutant library. After this mutant library, clone into expression plasmid and be transformed into impressionState bacterial isolates BL21.

Separate 1000 kinds of bacteriums and be inoculated in and comprise 500 μ l and add ampicillinsIn the Eppendorf pipe of LB culture medium. Sample is hatched 15 hours in shaking table. SecondMy god, by one or another kind of mensuration in the testing program of describing in the embodiment aboveThe isobutene amount producing.

Have after the clone of the isobutene amount significantly improving with identical testing program againInferior confirmation. Once confirm that this improves, extract plasmid order-checking from each improved clone.The sudden change that qualification is worked to improved activity is also combined described prominent on identical plasmidBecome. Comprise the different plasmids that improve sudden change and be transformed into again competence bacterium, and carry out identicalAnalysis.

The clone who comprises combinatorial mutagenesis is tool compared with only comprising the clone of independent improvement sudden changeThere is significantly more high activity, set it as afterwards basis for new round sudden change/screening, withQualification has the active mutant of further improvement.

After this testing program completes, select have the most highly active comprise several sudden changes gramGrand.

Embodiment 9: from the method for 3-hydracrylic acid synthesizing ethylene

The gene of the enzyme of describing in coding embodiment 1 is inserted can be Escherichia coli bacteriumIn strain, express the plasmid of recombinant protein. Plasmid is transformed into described bacterial isolates. TransformThere is propyl group diphosphonic acid (10mg/l) and 3-hydracrylic acid (1g/l) after bacteriumIn fermentation tank, cultivate. The existence of recombinase causes the conversion of 3-hydracrylic acid to ethene,It is naturally degassed and reclaimed by the gas extraction system that is positioned at fermentation tank top. Afterwards at gasIn body sample, use infrared detection in the strongest spectrum portion of ethene transmitting by gas chromatographyDivide and measure ethene.

Embodiment 10: from the method for 3-hydroxybutyrate synthesizing propylene

The gene of the enzyme of describing in the enzyme of describing in coding embodiment 1 or embodiment 4 is insertedEnter the plasmid that can express recombinant protein in coli strain. Plasmid is transformed into instituteState bacterial isolates. After the bacterium transforming, there is ethyl diphosphonic acid (10mg/l) and 3-In the fermentation tank of hydroxybutyric acid (1g/l) (Sigma, numbering 166898), cultivate. RestructuringThe existence of enzyme causes the conversion of 3-hydroxybutyrate to propylene, and it is naturally degassed and by being positioned atThe gas extraction system on fermentation tank top reclaims. In gaseous sample, pass through afterwards gas phase layerAnalyse by infrared detection at the strongest spectra part measurement propylene of propylene transmitting.

Embodiment 11: from the method for glucose synthesizing propylene

The gene of the enzyme of describing in the enzyme of describing in coding embodiment 1 or embodiment 4 is insertedEnter the plasmid that can express recombinant protein in alcaligenes eutrophus bacterium. Plasmid is transformed intoDescribed bacterial isolates.

After the bacterium transforming, there is the fermentation tank neutralization of glucose and ethyl diphosphonic acidUnder micro-aerobic condition, cultivate, experience afterwards heat shock to induce them to produce a large amount of 3-hydroxylsBase butyric acid. When causing 3-hydroxybutyrate to propylene, the existence of recombinase transforms, itsNaturally degassed and reclaimed by the gas extraction system that is positioned at fermentation tank top.

Embodiment 12: from the method for glucose synthesizing propylene

This embodiment has described the method closely similar with embodiment 11. The main distinction existsIn using the coli strain of modifying to prepare 3-hydroxybutyrate, and non-natural bacterial strain exampleAs alcaligenes eutrophus. Described bacterial strain obtains by the transformation of metabolic pathway, to produce 3-Gathering of hydroxybutyric acid. Add the MDP describing in for example embodiment 1 or embodiment 4Decarboxylase makes 3-hydroxybutyrate be converted into propylene.

Embodiment 13: from the method for the synthetic isobutene of glucose

The gene of the enzyme of describing in coding embodiment 1 is inserted can be Escherichia coli bacteriumIn strain, express the plasmid of recombinant protein, thereby described Escherichia coli thank in modification by the successive dynastiesHMB is synthesized in source.

After bacterium, in the fermentation tank that has glucose He under micro-aerobic condition, cultivate. HeavyThe existence of group enzyme transforms when causing HMB to isobutene, and it certainlySo degassed and reclaimed by the gas extraction system that is positioned at fermentation tank top.

Claims (33)

1. prepare the method for end alkene, it is characterized in that comprising with MDP decarboxylase conversion 3-hydroxyl alkaneAcid is the step of corresponding end alkene, and wherein 3-hydroxy alkanoic acid is following formula: compound:

HO-CO-CH₂-C(R₁)(R₂)-OH，

Wherein R₁And R₂Independently be selected from the alkyl of hydrogen atom and linearity or branch, described alkyl has1 to 4 carbon atom,

Wherein MDP decarboxylase is selected from the MDP decarboxylase shown in SEQIDNO:6-17 and 19.

2. according to the method for claim 1, it is characterized in that 2 substituting groups on 2 carbon of alkeneIn at least 1 or be the alkyl of linearity or branch.

3. according to the method for claim 1 or 2, it comprises the step that 3-hydroxybutyrate is converted into propyleneSuddenly.

4. according to the method for claim 1 or 2, it comprises 3-hydroxypentanoic acid is converted into 1-butyleneStep.

5. according to the method for claim 1 or 2, its comprise HMB is converted into differentThe step of butylene.

6. according to the method for claim 1 or 2, it comprises 3-hydroxy-3-methyl valeric acid is converted into differentThe step of amylene.

7. prepare the method for isobutene, it is characterized in that it comprises following steps: by MDP decarboxylationEnzymatic conversion HMB, wherein MDP decarboxylase is selected from SEQIDNO:6-17 and 19Shown MDP decarboxylase.

8. according to the method for claim 1 or 7, in reaction, add confactor according to described method.

9. method according to Claim 8, wherein said confactor is from phosphoric anhydride family, by leading toFormula R-O-PO₂H-O-PO₃H₂Represent.

10. according to the method for claim 9, wherein R is hydrogen atom or methyl, and also can be arbitrarilyLinear, branch or cyclic alkyl, or other unit price organic groups arbitrarily.

11. according to the method for claim 10, and wherein R is ethyl or propyl group.

12. methods according to Claim 8, wherein said confactor is from Medronate listEster, has general formula R-O-PO₂H-CH₂-PO₃H₂。

13. according to the method for claim 12, and wherein R is hydrogen atom or methyl, and also can be arbitrarilyLinear, branch or cyclic alkyl, or other unit price organic groups arbitrarily.

14. according to the method for claim 13, and wherein R is ethyl or propyl group.

15. according to the method for claim 1 or 7, transforms and exists at cosubstrate according to described methodUnder carry out.

16. according to the method for claim 15, and wherein said cosubstrate is the chemical combination that comprises phosphoric anhydrideThing.

17. according to the method for claim 16, and wherein said cosubstrate is ATP, rNTP, dNTPOr the mixture of several these molecules, polyphosphate or pyrophosphoric acid.

18. according to the method for claim 1 or 7, it is characterized in that step of converting is at cell free systemIn external carrying out.

19. according to the method for claim 1 or 7, it is characterized in that step of converting is described in excessively expressingThe microorganism of MDP decarboxylase carries out under existing.

20. according to the method for claim 1 or 7, it is characterized in that using having endogenous generation oneOr the natural or artificial performance of multiple 3-hydroxy alkanoic acid, and further express or cross express described natural orThe microorganism of modified MDP decarboxylase, thus directly prepare end alkene from carbon source.

21. according to the method for claim 20, and wherein microorganism is alcaligenes eutrophus (AlcaligenesOr the bacterium of Bacillus megatherium (Bacillusmegaterium) bacterial strain eutrophus).

22. according to the method for claim 21, and wherein microorganism is that dyed body is modified or plasmid transformsWith recombinant bacteria, yeast or the fungi of one or more 3-hydroxy alkanoic acids of excessive generation.

23. according to the method for claim 20, and wherein carbon source is glucose or other hexoses, wood arbitrarilySugar or arbitrarily other pentoses, glycerine or arbitrarily other polyalcohols or starch, cellulose, hemicellulose,Poly-3-hydroxy alkanoic acid or arbitrarily other polymer, described method is being selected from amylase, half fibre by useTieing up the suitable enzyme of plain enzyme, cellulase, poly--3-hydroxy alkanoic acid enzyme, is single by described depolymerizationUnder the system of body exists, and/or carry out under specific electrochemical conditions.

24. according to the method for claim 20, it is characterized in that using having endogenous generation one or manyPlant the natural or artificial performance of 3-hydroxy alkanoic acid, and further mistake is expressed natural or modified decarboxylasePhotosynthetic microorganism, thereby the CO existing from solution₂Directly prepare end alkene.

25. according to the method for claim 24, it is characterized in that described photosynthetic microorganism is that blue algae is thinBacterium or micro-algae.

26. according to the method for claim 1 or 7, it is characterized in that using allowing carbon source to be converted into 3-The first microorganism of hydroxy alkanoic acid, with use separate or by the decarboxylase of the second microbial expression,Make 3-hydroxy alkanoic acid be converted into end alkene.

27. according to the method for claim 1 or 7, it is characterized in that using expression MDP decarboxylasePlant or non-human animal, to prepare end alkene by the decarboxylation of 3-hydroxy alkanoic acid.

28. according to the method for claim 27, it is characterized in that multicellular organism is in some metabolic pathwayIn further modified, to synthesize one or more 3-hydroxy alkanoic acids.

29. according to the method for claim 1 or 7, and it comprises collects degassed end alkene from reactionThe step of gas.

30. according to the method for claim 1 or 7, it is characterized in that method enters under micro-aerobic conditionOK.

The microorganism of 31.MDP decarboxylase or generation MDP decarboxylase is last from the preparation of 3-hydroxy alkanoic acidPurposes in end ene compound, wherein 3-hydroxy alkanoic acid is following formula: compound:

HO-CO-CH₂-C(R₁)(R₂)-OH，

Wherein R₁And R₂Independently be selected from the alkyl of hydrogen atom and linearity or branch, described alkyl has1 to 4 carbon atom,

Wherein MDP decarboxylase is selected from the MDP decarboxylase shown in SEQIDNO:6-17 and 19.

32. compositions, its microorganism that comprises generation MDP decarboxylase, suitable culture medium and 3-Hydroxy alkanoic acid compound or can be the carbon source of 3-hydroxy alkanoic acid compound, wherein MDP by microbial conversionDecarboxylase is selected from the MDP decarboxylase shown in SEQIDNO:6-17 and 19.

33. have the natural or artificial performance of one or more 3-hydroxy alkanoic acids of endogenous generation, go forward side by side oneMulticellular organism or the microorganism of expressing natural or modified MDP decarboxylase are expressed or crossed to step, usesIn directly preparing end alkene from carbon source, wherein MDP decarboxylase is selected from SEQIDNO:6-17 and 19Shown MDP decarboxylase.